Cell migration is a fundamental property of most mammalian cells involved in the normal development of an organism, as well as in maintenance of the organism, for example, during healing of wounds or in the immune response system to infection. This migration process is also involved in the disease processes such as in the metastasis of cancer cells to establish secondary tumors and in angiogenesis by endothelial cells to support tumor growth. Accordingly, understanding cell migration and control of cell migration are important, and point to control of this behavior as a potential therapeutic target for cancer treatment.
Traditionally, wound healing assays have been employed in evaluating cell migration. Wound healing assays have been carried out in tissue culture for many years to monitor cell behavior, including estimating the migration and proliferative capacities of different cells and of cells, under different conditions.
These assays generally involve first growing cells to form a confluent monolayer. The monolayer is then wounded or disrupted by destroying or displacing a group of cells. Common methods for disrupting the cell monolayer comprise scratching a line (i.e., manually dragging a pointed device) through the layer with any of several different tools such as a needle, razor blade, plastic pipette tip or by removing a small area of cells, e.g., with a spinning circular pad. Additionally, wounding may be carried out using a very small spinning disk to give a more reproducible area to follow. Once a wound is achieved, the wound is then microscopically observed or photographed over time to assess the rate at which the damaged area is filled in by neighboring cells. Unfortunately, the above-noted wounding methods require extensive manipulation of the cultured cells, making these wounding methods expensive and, further more, difficult to accurately reproduce, and to verify experimental results.
As noted, after the disruption is accomplished, the area is inspected microscopically at different time intervals as the cells move in and fill the damaged area. This “healing” can take from several hours to over a day depending on the cell type, growing conditions, and the extent of the “wounded” region. The results may be presented by a series of photo-micrographs; or in more sophisticated measurements, the microscopic views may be subjected to image processing such that data is expressed in quantitative terms.
An alternative form of measuring cell behavior that replaced the commonly used microscopic observations utilizes electrical sensing. One example is disclosed in U.S. Pat. No. 5,187,096, which is hereby incorporated by reference and referred to herein as the “ECIS™ system”. Specifically, the ECIS™ system (Electric Cell-substrate Impedance Sensing) sold by Applied Biophysics, Inc., passively analyzes cell behavior by applying a weak AC current, and measuring voltage changes. The device can be used to monitor various cell behaviors, including morphology changes and cell motions in animal cells that attach and spread out and crawl on the bottom of tissue culture vessels. In the ECIS™ system, cells are grown upon, e.g., a small gold film electrode (e.g., 5×10−4 cm2) mounted to the bottom of a small well with (in one embodiment) a much larger counter electrode completing the circuit using a standard tissue culture medium as an electrolyte. A weak (e.g., approximately 1 microamp) AC current (usually in the frequency range from 100 to 40,00 Hz) is applied to the electrode. This small current results in a voltage drop across the small, active electrode of only a few millivolts. Voltage drops and currents this small do not affect the health of the cells.
Variations in the measured voltage comprise the measurement. As animal cells attach and spread upon the small, active electrode, they force the current to flow under and between the cells resulting in changes in impedance and hence, in the measured voltage across the electrodes. These changes can be followed and provide a non-invasive means to monitor changes in cell behavior. For example, using the measured voltages, one can infer cell morphology and cell movements, which are important research measurements that form the basis of many biomedical and biological assays.
While the ECIS™ system allows for automated and passive monitoring of cell behavior following a disruption, the traditional requirement of wounding the cell culture still remains.